Cd19 buv395

A panel of monoclonal antibodies was developed to enable phenotypic characterization of B and T lymphocyte subpopulations: CD3-FITC, CD4-APC, CD25-BV421, FoxP3-PE, CD19-PeCy7, CD9-BB700 (BD Bioscience, France), CD24-BV510, and CD38-APC-Cy7 (Sony Biotechnology, UK); hematopoietic stem and progenitor cells: CD34-BV421 (Biolegend, France), cKit-APC-H7, CD135-PE, lineage-FITC, Sca-1-BV510, CD16/32-PerCPCy5.5, CD127-APC (BD Bioscience), and TLR-4-PeCy7 (Biolegend); and dendritic cells: CD11c-PeCy7 (eBioscience, France), CMH-II-FITC, Siglec-H-BV510, CD11b-APC-H7, and CD103-PerCP5. For intracellular staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences). For in vitro human B cell differentiation experiments, cells were stained with Fixable Viability Dye eFluor 450 to identify dead cells, followed by staining with CD25-BV605 (Biolegend), CD19-BUV395, CD9-FITC, CD27-BUV737, CD38-BV711 and intracellular IL-10-PE (BD Bioscience). Cells were analyzed on a Canto II flow cytometer (BD Biosciences). Data were acquired using Diva 8.0 software and analyzed with FlowJo X (TreeStar, Williamson Way, Ashland, USA). Fluorescence minus one staining controls were used for all panels, and dead cells were removed using viability staining.

Bone marrow of Myce and littermate wild‐type control were harvested into single‐cell suspension with red cell lysis and stained with CD19‐Pacific Blue (1D3, BD, NJ, USA), B220‐PE cy7 (RA3‐6B2, BD), CD24‐APC (M1/69, in house), IgM‐FITC (331.12, in house), CD43‐PE (S7, BD). Spleen red cell lysed single cell suspensions were stained with CD19‐BB700 (1D3, BD), B220‐APC (Thermo Fisher, Waltham, USA), CD23‐PE cy7 (B3B4, Thermo Fisher), CD21‐BV421 (7E9, BioLegend, San Diego, USA), CD138‐PE (281–2, BD). The peritoneal cavity wash was stained with CD19‐PE (ID2, in house), B220‐APC (RA3‐6B2, Thermo Fisher), CD23‐PE cy7 (B3B4, Thermo Fisher), CD5‐BB700 (53–7.3, BD), CD11b‐eFlour450 (M1/70, Thermo Fisher). Lymph node and thymus of single cell suspensions were stained with CD19‐BUV395 (1D3, BD), (TCRβ‐PE (H57‐597, BD), CD4‐BV421 (GK1.5, BioLegend) and CD8a‐PerCp/Cy5.5 (53–6.7, eBioscience, San Diego, USA). Flow cytometry analysis was performed on a BD LSRFortessa X‐20 analyzer (BD).

The following antibodies were used to stain PCW cells: B220 PerCP-Cy5.5 (BioLegend, 103236), CD5 PE-Cy7 (BioLegend, 100622), CD19 BUV395 (BD Biosciences, 563557), IgM APC (Biolegend, 406509), PD-L2 BV421 (BD Biosciences, 564245), CD11b APC-Cy7 (BioLegend, 101226), Live/dead Ghost violet 510 (Tonbo Biosciences, 13-0870). For cells isolated from PZTD^+/+ mice, ZsGreen fluorescent protein was analyzed using the same channel as FITC. For cells isolated from CD19-Cre^+/- PTZD^+/+ mice, TdTomato fluorescent protein was analyzed using the same setting for TexasRed. To analyze TILs, CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), was added to the above panel replacing B220 for leukocyte gating. The stained samples were analyzed at the Boston University Medical Campus Flow Cytometry core facility using a BD LSRII flow cytometer and a Beckman Coulter MoFlo for cell sorting. Live leukocytes were gated by forward and side scatters, live/dead Ghost violet 510 and CD45 staining. B1 cells were gated as B220IntCD5Int population from the live leukocyte gate. L2pB1 cells were gated as PD-L2+IgM+ from the B1 cell gate. In the transgenic-knock in mice, L2pB1 cells were also identified by the expression of ZsGreen and TdTomato (Supplementary Figure S1).

GC responses were analyzed as previously reported (60 (link)): BALB/c mice were immunized with NE formulations containing 10 μg of eOD-GT8, 5 μg of 3M-052, and 0.1 μg of squalene oil. After 12 days, mesLNs were collected and single-cell suspensions were prepared. Cells were first incubated with live/dead stain (Zombie UV, BioLegend) at 1:750 dilution in 100 μl of PBS for 10 min at 25°C and then with antimouse CD16/32 antibody at 1:100 dilution in 100 μl of fluorescence-activated cell sorting buffer for 10 min at 25°C. Cells were stained against antimouse CD90.2 BV785 at 1:200 (clone 30-H12; BioLegend), CD19 BUV395 at 1:200 (1D3; BD Biosciences), CD38 FITC at 1:200 (90; BioLegend), GL7 PerCP-Cy5.5 at 1:150 (GL7; BioLegend), IgA AF647 at 1:100 (SouthernBiotech), eOD-tetramer BV605 at 1:100, and eOD-tetramer BV421 at 1:100 [tetramers formed by incubating 4 equivalents of biotinylated eOD with 1 equivalent of streptavidin-BV605 or streptavidin-BV421 (BioLegend) 18 hours at 4°C, prior to staining] in 100 μl of flow cytometry buffer for 20 min at 25°C. Cells were washed twice and fixed using 2% PFA. Data acquisition was performed using BD FACSymphony A3 and analyzed with FlowJo 10.

In vivo uptake studies were carried out following procedures we previously developed (60 (link)): BALB/c mice were immunized with NE formulations containing 10 μg of AF647-eOD-GT8, 5 μg of green fluorescent BODIPY FL C₁₂ cholesteryl ester, 5 μg of 3M-052, and 0.1 μg of squalene oil. Twenty-four hours later, mesLNs were collected and single-cell suspensions were prepared. Cells were first incubated with live/dead stain (Zombie UV, BioLegend) at 1:750 dilution in 100 μl of PBS for 15 min at 25°C and then with antimouse CD16/32 antibody (TruStain FcX, BioLegend) at 1:100 dilution in 50 μl of flow cytometry buffer for 10 min at 25°C. Cells were stained against antimouse CD3ε PE-CF594 (clone 145-2C11; BD Biosciences), CD19 BUV395 (1D3; BD Biosciences), CD11b BUV737 (M1/70; BD Biosciences), CD11c BV711 (N418, BioLegend), F4/80 PE (BM8; BioLegend), Ly6C BV785 (HK1.4, BioLegend), and Ly6G BV421 (1A8, BioLegend) at 1:100 dilution in 100 μl of flow cytometry buffer for 15 min at 25°C. Cells were washed twice and fixed using 2% PFA. Data acquisition was performed using BD FACSymphony A3 and analyzed with FlowJo 10.

Allergic lung inflammation was induced in Myce and wildtype mice using intraperitoneal ovalbumin (OVA)/alum sensitization followed by nebulized OVA challenge as described previously.
²² (link) On the day after the final challenge, ~200 μL of blood was collected into non‐heparinized tubes by retro‐orbital bleeding, and the mice were then killed by CO₂ inhalation after which BAL was performed (250 μL twice with sterile PBS). Flow cytometry was performed fresh on BAL cells using the following staining panel: CD19‐BUV395 Clone#1D3, CD8a‐PerCPeFluor710 Clone#52–6.7, SiglecF‐PE Clone#E50‐2440 (BD), Ly6c‐eFluor450 Clone#HK1.4, CD11c‐FITC Clone #N418, GR1‐PECy7 Clone#RB6‐8C5, TCRβ‐APCeFluor780 Clone #H57‐597 (eBioscience), CD4‐Alexa647 Clone#GK1.5, CD11b‐Alexa700 Clone#M1/70 from WEHI Antibody Facility (Supplementary table 4). Flow Cytometry was performed on a BD LSRFortessa X‐20 analyzer (BD). SYTOX Blue Dead Cell Stain was used to exclude dead cells from analysis and SPHERO Rainbow Beads (BD) were used to calculate absolute cell counts.